• 慢冻快融（慢——梯度）

3.1 Safely in Cell Culture

• Substances Hazardous 有危险的 to Health
• Waste Disposal
• Use of Cell Culture areas

3.2 Cell Culture Enemies

• Micro-organisms grow ~10-50 times faster than mammalian cells, which take ~8-16 hours to divide. They are more tolerant to variations in temperature, pH and nutrient supply than cells.
• Cells are most vulnerable脆弱的，易受伤的 to contamination when our aseptic technique is bad and the culture becomes infected with bugs.

—>This can lead to the development of antibiotic resistant micro-organisms.

• Never use contaminated受污染的 cells. They may not react in the same way as uncontaminated cells. Poor aseptic technique is the major cause of infections.
• Cell Culture Enemies

3.3 antiseptic technique

【preparation】 good protocols and schedule. Preparing materials and bring to culture room. Sterilizing.

【sterilizing】 spray 75％ethanol , UV 30-50min

【washing and coating】 UV 30min lab coat. Spray 75% to hands, gauze mask, hat.

【fire burning】ethanol乙醇、酒精 lamp, burning frequently

【operation】 quick in movement

3.4 Cell culture observation

１．Culture color and optical density ２．Cell growth state

Cell viability:

①Cell viability is determined by staining the cells with trypan blue. ②As trypan blue dye is permeable to non-viable cells or death cells whereas it is impermeable to this dye ③Stain the cells with trypan dye and load to haemocytometer血球计 and calculate % of viable cells ④% of viable cells= Nu. of unstained cells x 100 total nu. of cells

• Cell viability：0.4% trypan blue 1:1 cell mixing, 2-3min, death cell will be stained.